The authors would like to thank Dr. Meenhard Herlyn (Wistar Institute) and Dr. M. Celeste Simon (University of Pennsylvania) for providing reagents and technical support. We also thank the members of our laboratories for helpful discussion. This work was supported by the National Institutes of Health (NIH) grants (R01CA187718, R01CA148768 and R01CA142723), the NCI Cancer Center Support Grant (NCI P30 CA016520), and the Penn CFAR award (P30 AI 045008).

Human neonatal foreskins were obtained from Penn Skin Disease Research Center. Adult human fibroblasts were obtained from discarded normal skin after surgery. All the protocols were approved by the University of Pennsylvania Institutional Review Board.
1. Isolation of human dermal fibroblasts
<ol>
	<li>Use a pair of scissors to trim off fat and subcutaneous tissue from the human neonatal foreskin and cut the skin sample in halves or quarters.</li>
	<li>Incubate the tissue in 5 mL of 10 mg/mL dispase II in PBS supplemented with antibiotic-antimycotic at 4 &#176;C overnight.</li>
	<li>In a sterile 10 cm dish, carefully separate the epidermis from the dermal layers using microdissection forceps.</li>
	<li>Transfer the resulting dermal tissue to a 15 mL conical tube containing 5 mL of 2 mg/mL collagenase type IV in FBS-free medium supplemented with antibiotic-antimycotic.</li>
	<li>Incubate the sample at 37 &#176;C in 5% CO2 for 4-6 h with periodic shaking until just a few macroscopic tissue aggregates remain.</li>
	<li>Release single cells from the dermis by pipetting up and down (triturating) vigorously ten times with a 10 mL pipette.</li>
	<li>Centrifuge at 180 x g for 5 min, and discard the supernatant.</li>
	<li>Plate the dissociated cells in DMEM medium supplemented with 10% fetal calf serum, 1% non-essential amino acids, and 1% L-glutamine at 37 &#176;C in 5% CO2.</li>
</ol>
2. Recombinant MCPyV virion preparation
<ol>
	<li>Digest 50 &#181;g of pR17b plasmid (carrying MCPyV genome) with 250 U of BamHI-HF in a 200 &#181;L volume (4 h at 37 &#176;C) to separate the viral genome from the vector backbone (Figure 2).</li>
	<li>Add 1200 &#181;L of buffer PB (supplemented with 10 &#181;L of 3 M NaAc, pH 5.2) to the digested DNA and purify over 2 miniprep spin columns (20 &#181;g DNA capacity). Elute the digested pR17b plasmid from each column with 200 &#181;L of TE buffer (10 mM Tris-HCl, pH8.0, 1 mM EDTA).</li>
	<li>Prepare the ligation reaction in a 50 mL centrifuge tube. Add 400 &#181;L of purified plasmid DNA from step 2.2, 8.6 mL of 1.05x T4 ligase buffer and 6 &#181;L of high concentration T4 ligase. Incubate at 16 &#176;C overnight.</li>
	<li>Add 45 mL of buffer PB (supplemented with 10 &#181;L of 3 M NaAc, pH 5.2) to the ligation and use a vacuum manifold to load through 2 miniprep spin columns. Elute each column with 50 &#181;L of TE buffer. Expect a yield of about 30 &#181;g of DNA.</li>
	<li>In the late afternoon/evening, seed 6 x 106 293TT28 cells into a 10 cm dish containing DMEM medium supplemented with 10% fetal calf serum, 1% non-essential amino acids, and 1% L-glutamine without hygromycin B.</li>
	<li>The next morning, ensure that the cells are about 50% confluent. Transfect using 66 &#181;L of transfection reagent (1.1 &#181;L/cm2), 12 &#181;g of re-ligated MCPyV isolate R17b DNA from step 2.4, 8.4 &#181;g of ST expression plasmid pMtB and 9.6 &#181;g of LT expression plasmid pADL*29.</li>
	<li>When the transfected cells are nearly confluent, the following day, trypsinize the cells and transfer them to a 15 cm dish for continued expansion.</li>
	<li>Optionally take a small number of 293TT cells upon expansion and perform IF staining for MCPyV LT (CM2B4) and VP1 (MCV VP1 rabbit30) to determine transfection efficiency. At this stage, nuclear LT signal may be visible but VP1 expression probably will not be detectable.</li>
	<li>When the 15 cm dish becomes nearly confluent (usually 5-6 days after initial transfection), transfer the cells into three 15 cm dishes. Harvest the cells from the 15 cm dishes when they become nearly confluent and follow the virus harvest protocol below. 
	NOTE: Optionally perform quality control IF as described in step 2.8. Most of the cells should be both MCPyV LT and VP1 positive at this stage.</li>
	<li>To harvest the virus, trypsinize cells, spin at 180 x g for 5 min at RT and remove the supernatant. Add one cell pellet volume of DPBS-Mg (DPBS with 9.5 mM MgCl2 and 1x antibiotic-antimycotic). Then add 25 mM ammonium sulfate (from a 1 M pH 9 stock solution) followed by 0.5% Triton X-100 (from a 10% stock solution), 0.1% Benzonase and 0.1% of an ATP-Dependent DNase. Mix well and incubate at 37 &#176;C overnight.</li>
	<li>Incubate the mixture for 15 min on ice and then add 0.17 volume of 5M NaCl. Mix and incubate on ice for another 15 min. Spin for 10 min at 12,000 x g in a 4 &#176;C centrifuge. If the supernatant is not clear, gently invert the tube and repeat the spinning step. Transfer the supernatant to a new tube.</li>
	<li>Resuspend the pellet using one volume of DPBS supplemented with 0.8 M NaCl, and spin again, as described in step 2.11.</li>
	<li>Combine the supernatants from the step 2.11 and 2.12, and spin one more time as described in step 2.11.</li>
	<li>Pour gradients of iodixanol in thin wall 5 mL polyallomer tubes by underlaying (27%, then 33%, then 39%) ~0.7 mL steps using a 3 mL syringe fitted with a long needle or a p1000 pipette.</li>
	<li>Load 3 mL of clarified virus-containing supernatant on the prepared iodixanol gradient.</li>
	<li>Spin for 3.5 h at 234,000 x g and 16 &#176;C in an SW55ti rotor. Set the acceleration and deceleration to slow.</li>
	<li>After ultracentrifugation, collect 12 fractions in siliconized tubes (each fraction is ~400 &#181;L).</li>
	<li>Analyze the fractions for the presence of virus by dsDNA reagent and/or Western blot for VP1 (MCV VP1 rabbit30). Pool gradient fractions with peak dsDNA and/or VP1 content and characterize the stock by quantitative PCR to calculate the viral genome equivalent31. Store MCPyV virion stock at -80 &#176;C.</li>
</ol>
3. Infection
<ol>
	<li>Maintain primary human dermal fibroblasts in DMEM with 10% fetal calf serum, 1% non-essential amino acids and 1% L-glutamine. Upon reaching confluence, split fibroblasts 1:4 without spinning down. 
	NOTE: For highest MCPyV infection efficiency, use primary fibroblasts between passages 5 and 12 that are actively dividing at the time of plating.</li>
	<li>To infect human dermal fibroblasts, aspirate the medium and wash the cells with DPBS.</li>
	<li>Add 1 mL of 0.05% Trypsin-EDTA to the dish and incubate at 37 &#176;C for 5-10 min.</li>
	<li>Check under the microscope to make sure that the cells are coming off the dish.</li>
	<li>Add 10 mL of DMEM/F12 medium containing 20 ng/mL EGF, 20 ng/mL bFGF and 3 &#181;M CHIR99021, all of which stimulate MCPyV infection24, to the dish and transfer the cell solution into a 15 mL tube.</li>
	<li>Spin down the cells at 180 x g for 2 min, and discard the supernatant. Resuspend the cells in DMEM/F12 medium containing 20 ng/mL EGF, 20 ng/mL bFGF and 3&#181;M CHIR99021 at 2-4 x 104 cells per mL.</li>
	<li>Seed 200 &#181;L of the cell suspension supplemented with 1 mg/mL collagenase type IV into each well of a 96-well plate. Thaw MCPyV virion stock on ice. Add 109 viral genome equivalents of MCPyV virions (calculated in step 2.18) per 1 &#181;L of iodixanol for each 2,500 to 5000 cells to be infected.&#160;&#160;Tap the side of the plate gently and place the plate in the incubator.</li>
	<li>After incubation at 37 &#176;C in 5% CO2 for 48-72 h, add 20% FBS to each well.</li>
	<li>Allow the infection to proceed at 37 &#176;C in 5% CO2 for 72-96 h.</li>
</ol>
4. Immunofluorescent staining
<ol>
	<li>Fix cells obtained in step 3.9 with 3% paraformaldehyde in PBS for 20 min.</li>
	<li>Wash the cells with PBS twice.</li>
	<li>Incubate the cells in blocking/permeabilization buffer (0.5% Triton X-100, 3% BSA in PBS filtered through 0.22 &#181;m filter) at RT for 1 h.</li>
	<li>Incubate the cells with the following primary antibodies: mouse monoclonal anti-MCPyV LT (CM2B4) (1:1000) and rabbit polyclonal anti-MCPyV VP1 antibody (1:2000), at RT for 3 h.</li>
	<li>Wash the cells with PBS three times.</li>
	<li>Incubate the cells with the secondary antibodies: Fluor 594 goat anti-mouse IgG (1:1000) and Fluor 488 goat anti-rabbit IgG (1:500) at RT for 1 h.</li>
	<li>Wash cells with PBS three times.</li>
	<li>Counterstain the cells with 0.5 &#181;g/mL DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride).</li>
	<li>Mount coverslips on glass slides and analyze using an inverted fluorescence microscope.</li>
</ol>
5. In situ DNA-HCR
NOTE: This technique requires that cells be seeded on coverslips. For this purpose, the infection conditions described above (step 3) may be scaled up to the 24-well plate format.
<ol>
	<li>Fix cells cultured on coverslips with 4% PFA in PBS for 10 min. Wash the coverslips twice with PBS, then treat them with 70% ethanol to permeabilize the cells at 4 &#176;C overnight. 
	NOTE: Fixed samples can remain in this state for several days.</li>
	<li>Pre-hybridize samples by replacing the ethanol with probe hybridization buffer and incubating for 60 min at RT.</li>
	<li>Dilute the probe(s) (1 &#181;M) (Table 1) in probe hybridization buffer solution at 1:500 dilution 30 min prior to the end of the pre-hybridization step and incubate at 45 &#176;C.</li>
	<li>At the end of the incubations, pipette ~10 &#956;L of the diluted probe mix per coverslip on microscope slides. Place the coverslips, cell-side down, on their respective droplets of hybridization probe mix. Seal the edges and backs of the coverslips to the slides with a liberal amount of rubber cement.</li>
	<li>Heat the slides with added probes at 94 &#176;C for 3 min by setting the slides on the flat side of a heat block. Transfer the slides to a humidified chamber and incubate at 45 &#176;C overnight. To make a simple humidified chamber, lightly dampen sterile paper towels with dH2O and place in the bottom of a plastic container that seals with a rubber gasket. 
	NOTE: During heating the rubber cement can bubble briefly. However, ensure that the seal does not break.</li>
	<li>Carefully peel away the rubber cement with forceps and place the coverslips cell-side up back into wells of a 24-well plate. Wash the coverslips with probe wash buffer at RT three times.</li>
	<li>Incubate the coverslip in the amplification buffer (200 &#181;L in 24-well plates) at RT 30-60 min.</li>
	<li>Meanwhile, anneal each of the two labeled oligonucleotide hairpins recognizing the probes (Table 1) in separate PCR tubes by heating to 94&#160;&#176;C for 90 s and cooling to RT for 30 min while protecting from light. Mix the two hairpins in amplification buffer, each at a dilution of 1:50.</li>
	<li>Make a surface for the amplification reaction by stretching paraffin film over the open face of a 24-well plate lid. Pipette 50-100 &#181;L droplets of the hairpin/amplification buffer mixture onto the paraffin film for each coverslip. Use forceps to carefully remove the coverslips from the pre-amplification solution. Dry the coverslip by touching the edge to a porous disposable wipe, and place cell-side down onto the amplification droplet.</li>
	<li>Place the plate lid holding the coverslips into a humidified chamber and incubate overnight at RT in the dark.</li>
	<li>Return the coverslips to wells once more. Incubate the samples with 5x SSCT [5x sodium chloride sodium citrate (SSC) 0.1% Tween 20 filtered through a 0.22 &#181;m filter] containing 0.5 &#181;g/mL DAPI for 1 h at RT. Wash the samples twice with 5x SSCT at RT.</li>
	<li>Mount the coverslips on microscope slides. Analyze the cells and image the samples using an inverted fluorescence microscope.</li>
</ol>

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The authors have nothing to disclose.

The methods described above , including isolation of dermal fibroblasts from human skin tissue, preparation of recombinant MCPyV virions, infection of cultured cells, immunofluorescent staining, and a sensitive FISH method adapted from HCR technology, which should enable researchers to analyze MCPyV infection27. One of the most critical steps to achieving MCPyV infection in vitro is the production of high-titer virion preparations. Using the protocol for preparation of recombinant MCPyV virions de...

Merkel cell polyomavirus (MCPyV) is a small, double-stranded DNA virus that has been associated with a rare but aggressive skin cancer, Merkel cell carcinoma (MCC)1,2. The mortality rate of MCC, around 33%, exceeds that of melanoma3,4. MCPyV has a circular genome of ~5 kb1,5 bisected by a non-coding regulatory region (NCRR) into early and late coding regions1. The NCRR contains the viral origin of replication (Ori) and bidirectional promoters for viral transcription6,7. The early region encodes tumor antigen proteins called large T (LT), small T (sT), 57kT, alternative LT ORF (ALTO), as well as an autoregulatory miRNA1,8,9,10. The late region encodes the capsid proteins VP1 and VP211,12,13. LT and sT are the best-studied MCPyV proteins and have been shown to support the viral DNA replication and MCPyV-induced tumorigenesis5. Clonal integration of MCPyV DNA into the host genome, which has been observed in up to 80% of MCCs, is likely a causal factor for virus-positive tumor development14,15.
The incidence of MCC has tripled over the past twenty years16. Asymptomatic MCPyV infection is also widespread in the general population17,18,19. With the increasing number of MCC diagnoses and the high prevalence of MCPyV infection, there is a need to improve our understanding of the virus and its oncogenic potential. However, many aspects of MCPyV biology and oncogenic mechanisms remain poorly understood20. This is largely because MCPyV replicates poorly in established cell lines11,12,21,22,23 and, until recently, skin cells capable of supporting MCPyV infection had not been discovered22. Mechanistic studies to fully investigate MCPyV and its interaction with host cells have been hampered by a lack of cell culture system for propagating the virus5.
We discovered that primary human dermal fibroblasts (HDFs) isolated from neonatal human foreskin support robust MCPyV infection both in vitro and ex vivo24. From this study, we established the first cell culture infection model for MCPyV24. Building on this model system, we showed that the induction of matrix metalloproteinase (MMP) genes by the WNT/&#946;-catenin signaling pathway and other growth factors stimulates MCPyV infection. Moreover, we found that the FDA-approved MEK antagonist trametinib effectively inhibits MCPyV infection5,25. From these studies, we also established a set of protocols for isolating human dermal fibroblasts24,25, preparing MCPyV virions11,12, performing MCPyV infection on human dermal fibroblasts24,25 and detecting MCPyV proteins by IF staining26. In addition, we adapted the in situ DNA hybridization chain reaction (HCR) technology27 to develop a highly sensitive FISH technique (HCR-DNA FISH) for detecting MCPyV DNA in infected human skin cells. These new methods will be useful for studying the infectious cycle of MCPyV as well as the cellular response to MCPyV infection. The natural host reservoir cells that maintain MCPyV infection and the cells that give rise to MCC tumors remain unknown. The techniques we describe in this manuscript could be applied to examine various types of human cells to identify both the reservoir cells and origin of MCC tumors. Our established methods, such as HCR-DNA FISH, could also be employed in the detection of other DNA tumor viruses and the characterization of host cell interactions.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Fetal calf serum</td>
			<td style="width:185px;">HyClone</td>
			<td style="width:119px;">SH30071.03</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">MEM Non-Essential Amino Acids Solution, 100X</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">GLUTAMAX I, 100X</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td>L-Glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DPBS, no calcium, no magnesium</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td>14190136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">0.05% Trypsin-EDTA</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DMEM/F12 medium</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Recombinant Human EGF Protein, CF</td>
			<td style="width:185px;">R&#38;D systems</td>
			<td>236-EG-200</td>
			<td>Store at -80 degree celsius</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">CHIR99021</td>
			<td style="width:185px;">Cayman Chemical</td>
			<td>13122</td>
			<td>Store at -80 degree celsius</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">CHIR99021</td>
			<td style="width:185px;">Sigma</td>
			<td>SML1046</td>
			<td>Store at -80 degree celsius</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Collagenase type IV</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td>17104019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Dispase II</td>
			<td style="width:185px;">Roche</td>
			<td>4942078001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Antibiotic-Antimycotic</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td>15240-062</td>
			<td>Protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DMEM medium</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td>11965084</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Alexa Fluor 594 goat anti-mouse IgG</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td>A11032</td>
			<td>Protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Alexa Fluor 488 goat anti-rabbit IgG</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td>A11034</td>
			<td>Protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">OptiPrep Density Gradient Medium</td>
			<td style="width:185px;">Sigma</td>
			<td>D1556</td>
			<td>Protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Paraformaldehyde</td>
			<td style="width:185px;">Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">anti-MCPyV LT (CM2B4)</td>
			<td style="width:185px;">Santa Cruz</td>
			<td>sc-136172</td>
			<td>Lot # B2717</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">MCV VP1 rabbit</td>
			<td style="width:185px;"></td>
			<td>Rabbit polyclonal serum #10965</td>
			<td>https://home.ccr.cancer.gov/lco/BuckLabAntibodies.htm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Hygromycin</td>
			<td style="width:185px;">Roche</td>
			<td>10843555001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">Basic Fibroblast Growth Factors (bFGF), Human Recombinant</td>
			<td style="width:185px;">Corning</td>
			<td>354060</td>
			<td>Store at -80 degree celsius</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Benzonase Nuclease</td>
			<td style="width:185px;">Sigma</td>
			<td>E8263</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Plasmid-Safe ATP-Dependent DNase</td>
			<td style="width:185px;">EPICENTRE</td>
			<td>E3101K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Probe hybridization buffer</td>
			<td style="width:185px;">Molecular technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:339px;">Probe wash buffer</td>
			<td style="width:185px;">Molecular technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Amplification buffer</td>
			<td style="width:185px;">Molecular technologies</td>
			<td style="width:119px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Alexa 594-labeled hairpins</td>
			<td style="width:185px;">Molecular technologies</td>
			<td>B4</td>
			<td>Protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Triton X-100</td>
			<td style="width:185px;">Sigma</td>
			<td style="width:119px;">X100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Quant-iT PicoGreen dsDNA Reagent</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">P7581</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">BamHI-HF</td>
			<td style="width:185px;">NEB</td>
			<td style="width:119px;">R3136</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Buffer PB</td>
			<td style="width:185px;">Qiagen</td>
			<td style="width:119px;">19066</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">blue miniprep spin column</td>
			<td style="width:185px;">Qiagen</td>
			<td style="width:119px;">27104</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">50mL Conical Centrifuge Tubes</td>
			<td style="width:185px;">Corning</td>
			<td style="width:119px;">352070</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">T4 ligase</td>
			<td style="width:185px;">NEB</td>
			<td style="width:119px;">M0202T</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MagicMark XP</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">LC5602</td>
			<td></td>
		</tr>
	</tbody>
</table>

merkel cell polyomavirus infection and detection

Merkel cell polyomavirus (MCPyV) infection can lead to Merkel cell carcinoma (MCC), a highly aggressive form of skin cancer. Mechanistic studies to fully investigate MCPyV molecular biology and oncogenic mechanisms have been hampered by a lack of adequate cell culture models. Here, we describe a set of protocols for performing and detecting MCPyV infection of primary human skin cells. The protocols describe the isolation of human dermal fibroblasts, preparation of recombinant MCPyV virions, and detection of virus infection by both immunofluorescent (IF) staining and in situ DNA-hybridization chain reaction (HCR), which is a highly sensitive fluorescence in situ hybridization (FISH) approach. The protocols herein can be adapted by interested researchers to identify other cell types or cell lines that support MCPyV infection. The described FISH approach could also be adapted for detecting low levels of viral DNAs present in the infected human skin.

The protocol described in this manuscript allowed isolation of a nearly homogenous population of HDFs (Figure 1). As demonstrated by immunofluorescent staining, almost 100% of the human dermal cells isolated using the conditions described in this protocol were positively stained for dermal fibroblast markers, vimentin, and collagen I24, but negative for human foreskin keratinocyte marker K14 (Figure 1). <s...

Watch this Scientific Journal Video about Merkel Cell Polyomavirus Infection and Detection at JoVE.com

Merkel Cell Polyomavirus Infection and Detection

Here, we present a protocol to infect primary human dermal fibroblast with MCPyV. The protocol includes isolation of dermal fibroblasts, preparation of MCPyV virions, virus infection, immunofluorescence staining, and fluorescence in situ hybridization. This protocol can be extended for characterizing MCPyV-host interactions and discovering other cell types infectable by MCPyV.

Merkel cell polyomavirus (MCPyV) infection can lead to Merkel cell carcinoma (MCC), a highly aggressive form of skin cancer. Mechanistic studies to fully ...

merkel-cell-polyomavirus-infection-and-detection

Research

JoVE Journal

Immunology and Infection

9.8K Views.  University of Pennsylvania. Here, we present a protocol to infect primary human dermal fibroblast with MCPyV. The protocol includes isolation of dermal fibroblasts, preparation of MCPyV virions, virus infection, immunofluorescence staining, and fluorescence in situ hybridization. This protocol can be extended for characterizing MCPyV-host interactions and discovering other cell types infectable by MCPyV.

Video: Merkel Cell Polyomavirus Infection and Detection

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the detection of biomarkers, contaminants, and other molecules of interest. A commonly used technique for this purpose is the Enzyme-linked Immunosorbent Assay (ELISA), where often one antibody is directed towards a specific target molecule, and a second labeled antibody is used for the detection of the primary antibody, allowing for the absolute quantification of the biomolecule under study. However, the usage of antibodies as recognition elements limits the robustness of the method; as does the need of using labeled molecules. To overcome these limitations, molecular imprinting has been implemented, creating artificial recognition sites complementary to the template molecule, and obsoleting the necessity of using antibodies for initial binding. Further, for even higher sensitivity, the secondary labeled antibody can be replaced by biosensors relying on the capacitance for the quantification of the target molecule. In this protocol, we describe a method to rapidly and label-free detect and quantitate low-abundant biomolecules (proteins and viruses) in complex samples, with a sensitivity that is significantly better than commonly used detection systems such as the ELISA. This is all mediated by molecular imprinting in combination with a capacitance biosensor.

Here, we present a protocol for the detection and quantification of low abundant molecules in complex solutions using molecular imprinting in combination with a capacitance biosensor.

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...